Project Summary: The majority of proteins essential for mitochondrial functions are encoded in the nuclear DNA and subsequently imported from the cytosol by translocases of the mitochondrial outer and inner membranes, termed TOM and TIM, respectively. These translocases are multimeric protein complexes, which are extensively studied in fungi and later in humans and plants. However, mitochondrial protein import machinery has been poorly characterized in trypanosomatids, a group of ancient and early branching unicellular eukaryotes that possesses a single mitochondrion per cell. Recent studies by our laboratory and others identified a number of non-canonical protein components for this essential cellular process in Trypanosoma brucei. As part of this machinery, we previously characterized TbTim17, which is critical for mitochondrial protein import and thus essential for parasite survival in two major developmental forms, the procyclic and bloodstream forms, found in the insect vector and the mammalian bloodstream, respectively. TbTim17 is present in multiple protein complexes within the range of 300-1100 kDa. Using pull down assays we identified one relatively conserved protein, Tim50, and several other novel proteins. Among these, we found TbTim62, a non-canonical Tim protein, act as a critical assembly factor of the TbTim17 protein complexes and have a major effect on TbTim17 stability. In addition, we found that Tim50 interacts with TbTim17 and is involved in the import of N-terminal signal containing mitochondrial proteins. Besides, TbTim17 also associates with a group soluble small TbTims. In contrast to the two TIM complexes (TIM23 and TIM22) with different substrate specificities in fungi to mammals, trypanosomatids most likely possess a single TIM (TbTIM17) that is capable of importing various types of mitochondrial proteins. Based on these results we hypothesized that TbTim17 forms an evolutionarily divergent and modular type TIM complex by association with novel TbTims and imports different types of substrate protein into mitochondria. We will test this hypothesis by three aims; 1) to define the structural components of the TbTim17 protein complex; 2) to determine the unique and interactive functions of TbTim17, TbTim62, TbTim50, TbTim54, and small TbTims in T. brucei, and 3) to determine the structural domains of TbTims critical for their functions. We will use many conventional and specialized techniques to evaluate the properties of these TbTims, including their substrate specificities, ability to assemble into the TIM complex, role in the stability of this complex, protein-protein interactions, and unique structure-function relations. Together, these studies will elucidate the subunit composition and function of the divergent TbTIM complex in T. brucei that can also be extrapolated for other trypanosomatid parasites and thus illuminate the mechanism of mitochondrial protein import in a group of the earliest eukaryotes.